Studies on individuals and animals with genetic variants in serotonin function can shed light on the role of this neurotransmitter in behavior including the role of functional variants in serotonin genes in predisposing individuals to psychopathologies and to alcoholism. We are emphasizing the role of functional [candidate] alleles in behavior. Two 5-HT1A variants are rare amino acid substitutions (Gly22Ser and Val28Ile), one conservative and one nonconservative. The 5-HT2C variant is a common (allele frequency=0.18) nonconservative substitution (Cys23Ser). Two 5HT2A amino acid substitutions (Ala477Val and His452Tyr) have allele frequencies of 0.01 and 0.09. Rare serotonin transporter and 5-HT7 amino acid substitutions were also discovered. Three of these amino acid substitutions were shown to alter the functional properties of the corresponding receptor. 5HT1A Gly22Ser when expressed in CHO-K1 cells dramatically altered desensitization and down regulation of these receptors. 5HT2C Cys23Ser in oocytes and COS-7 cells decreased ligand binding. 5HT2A His452Tyr impaired signal transduction in platelets from subjects with the 452Tyr allele. For association and direct gene analysis, we have collected more than 40 cell lines from each of the following populations: anorexia nervosa (collaboratively with W. Kaye), obsessive compulsive disorder (D. Murphy), low CSF 5-HIAA with Type II alcoholism (M. Linnoila, M. Virkkunen, M. Eggert), and seasonal affective disorder (N. Rosenthal, N. Ozaki). For direct gene analysis, we mainly use dHPLC analysis and direct sequencing. Association of a TPH IVS-7 polymorphism with suicidality in impulsive alcoholic Finns was replicated. Also, series of promoter polymorphisms were detected and these polymorphisms and the IVS-7 polymorphism were evaluated for functionality [Rotondo et al]. Recently a new TPH gene, TPH2, was detected and this gene encodes a brain-specific TPH. By resequencing, we identified an uncommon amino acid substitution in TPH2 and a large panel of SNPs, and are performing haplotype-based linkage analysis of the gene in several datasets with anxiety, impulsivity and alcoholism phenotypes. Sib-pair linkage of 5HT1B, the terminal autoreceptor for serotonin neurons] to antisocial alcoholism was found in Finns and replicated in Southwestern American Indians [Lappalainen et al]. In a series of publications, we have shown that the 5HT2A-1438 G>A promoter variant is linked to anxiety related conditions. These include OCD, seasonal affective disorder, anorexia nervosa and anxiety-related scales from the Tridimensional Personality Questionnaire. A 5HT5 amino acid substitution was detected and reveals a preliminary linkage signal to schizophrenia [Iwata et al]. The serotonin transporter (5-HTT) plays a critical role in the termination of serotonergic neurotransmission by Na-dependent uptake of serotonin by the presynaptic neuron. 5-HTT also represents the initial site of action of certain antidepressant drugs and neurotoxins. A functionally significant polymorphism in the 5-HTT promoter was identified (HTTLPR). The polymorphism affects in vitro 5-HTT transcription and, ultimately, 5-HTT function. We linked HTTLPR to the two anxiety related subscales of the TPQ in a sib pair analysis (C. Mazzanti), replicating the earlier finding by Lesch and colleagues. The variant was also linked to SAD [Enoch et al], alcohol response [Schuckit et al], and BPRS psychoticsm rating in schizophrenia [Malhotra et al]. The serotonin transporter polymorphism was next linked to two intermediate phenotypes in the causal chain connecting functional gene variant to behavior. These were midbrain serotonin transporter density determined by B-CIT SPECT [Heinz et al] and amygdala metabolic activity following a cognitive fear challenge [Hariri et al]. A rare serotonin transporter missense variant was discovered in LNG, shown to be functional by NIMH colleagues, and studied in two families by collaborators in NIMH and at Western Psychiatric Institute. In these families, the HTT variant predicts a more severe behavioral syndrome which may include obsessionality, anxiety, and overt Asperger's syndrome.